L4: Nucleic Acid Structure and Detection Flashcards
What is a nucleic acid strucutre
- nitrogenous base unit: purine of pyrimidine
- 5B sugar unit: ribose or deoxyribose
- phosphate group
chargaff’s Rule
total Purine (AG) = total prymidine (TC)
Who predicted the d helix strucutre
- Watson and Crick, complementary bases held together by hydrogen bonds
How does base pairing work in DNA
- 2 hydrogen bonds for AT
- 3 hydrogen bonds for GC
- sugar/phosphate backbones are antiparallel ( 3’aligned with 5’)
What are the three d helix conformations
A- DNA short and wide right handed
B-DNA common
Z- left handed
Briefly go through chromosomes
- genome of species
human: 22+ X/Y
barley = 7
homologous chromomes: 2n or 1n cells
structure: nucelosomes (proteins + DNA), arms, centromere, telomere
One chromosome =
one DNA molecule
What are the elements of nuclear gene
- Distal Regulatory
- proximon elements
= promoter region
Transcriptional start sitre
Exon + Intron + Exon
= structural region
Transciptional termination site
Define Intron and Exon
Intron: intervening sections of non coding sequencing removed during processing of primary transcript
Exon: sequence from primary transcript, coding
What are restriction enzymes / endonucleases
- recognise specific nucleotides and produce a cut in ds DNA
- recongise 4-8 nucleotides usually 4pb, 6 is medium and 8 is rare
- palindormic
Name the applications
T4 DNA ligase
T4 RNA ligase
Taq Poly
Phosphatase
T4 Polynucloetide Kinase
DNAse 1
MMLV Reverse
AMV Reverse
Proteinase K
Ribonuclease A
Nuclease S1
Mung Bean Nuc
T4 DNA Ligase: cloning
T4 RNA ligase: RNA labeling, primer extension
Taq DNA Polymerase: PCR from DNA template
Phosphatase: prevent recircularization of DNA vectors
T4 Polynucleotide Kinase: Nucleic acid labeling
DNase I: elimination of DNA in RNA or protein
preparations
M-MLV Reverse Transcriptase: RT-PCR for long transcripts
AMV-Reverse Transcriptase Synthesis: of cDNA from RNA (RT-PCR)
Proteinase K: Removes proteins from DNA samples
Ribonuclease A: RNA elimination from DNA preparation
Nuclease S1: Degrades RNA or single-stranded DNA into 5′ mononucleotides
Mung Bean Nuclease: Degrades single stranded DNA and RNA
Difference between EcoRI and EcoRV
- EcoRI: produces sticky ends
- EcoRV: produces blunt ends
Name two blotting techniques and define them
Southern Hybridisation: binding radioactive/fluro DNA probe to DNA molecule immobilized on membrane filter (DNA by DNA)
Northern Hybridisation: “”” DNA probe between complementary bases in RNA and DNA probe
How do you determine the optimum temperature for the probe
Tm = 4(G+C) + 2(A+T) assumed 0.9M
What is UV Absorabce Quanitifaction based on
sample aborbance of light wat wavelength 260, due to aromatic nitrogen bases, related to nucleic acids
What are the general approximation of UV absorbance quantification
If A260
1 ds DNA = 60
ssDNA = 40
oligo = 30
equi molar amounts: 2:1
How does detection take place + details
visualisation of NA after seperations
- use small amounts of DNA/RNA but need good estimate of amount of DNA/RNA
use dyes like EtBr, prop Idone, SYBR green, RedSafe
How does gel electrophoreisis work
- seperate by length and size
- apply electric field and neg molecules move through argose
- short molecules travel farther
- proteins seperated by charge becaus eof the pores are too big to sieve proteins
